Autosomal recessive non‐syndromic hearing loss genes in Pakistan during the previous three decades

Abstract Hearing loss is a clinically and genetically heterogeneous disorder, with over 148 genes and 170 loci associated with its pathogenesis. The spectrum and frequency of causal variants vary across different genetic ancestries and are more prevalent in populations that practice consanguineous marriages. Pakistan has a rich history of autosomal recessive gene discovery related to non‐syndromic hearing loss. Since the first linkage analysis with a Pakistani family that led to the mapping of the DFNB1 locus on chromosome 13, 51 genes associated with this disorder have been identified in this population. Among these, 13 of the most prevalent genes, namely CDH23, CIB2, CLDN14, GJB2, HGF, MARVELD2, MYO7A, MYO15A, MSRB3, OTOF, SLC26A4, TMC1 and TMPRSS3, account for more than half of all cases of profound hearing loss, while the prevalence of other genes is less than 2% individually. In this review, we discuss the most common autosomal recessive non‐syndromic hearing loss genes in Pakistani individuals as well as the genetic mapping and sequencing approaches used to discover them. Furthermore, we identified enriched gene ontology terms and common pathways involved in these 51 autosomal recessive non‐syndromic hearing loss genes to gain a better understanding of the underlying mechanisms. Establishing a molecular understanding of the disorder may aid in reducing its future prevalence by enabling timely diagnostics and genetic counselling, leading to more effective clinical management and treatments of hearing loss.


| INTRODUC TI ON
Pakistan, home to a population of 232 million according to the February 2023 United Nations census, ranks as the fifth most populous country in the world.The country's population is dispersed across multiple provinces, each with unique ethnic and linguistic diversity backgrounds.Consanguineous marriage is prevalent in most communities, making Pakistan one of the countries with the highest consanguinity rates globally.Consequently, homozygous pathogenic alleles steadily accumulate, leading to an ultimate increase in the prevalence of autosomal recessive disorders such as congenital hearing loss (HL).Out of the estimated 14.5 million Pakistanis living with HL, about half have a genetic aetiology. 1According to the World Health Organization, more than 1.5 billion people around the world are impacted by HL, and roughly 28% of them, or 430 million individuals, have a disabling level of HL.This is concerning because the number of people with disabling HL is projected to exceed 700 million by 2050 (https:// www.who.int/ healt h-topics/ heari ng-loss).
Although individuals from the Pakistani community represent a minority of patients tested within the Asian population, 2 their contributions have yielded valuable insights into the molecular genetics and biology of hearing and deafness that hold relevance across diverse global genetic backgrounds.While this review does not aim to provide an exhaustive catalogue of all the variants identified in the Pakistani population, its goal is to highlight the exciting discoveries that have enriched our current understanding of the genetic epidemiology of HL.Although many genes have been implicated in HL (with many more yet to be discovered) in the Pakistani population, a dozen or so genes appear to play a predominant role in its genetic makeup.In this review, we provide a brief overview of the clinical heterogeneity and history of HL gene discovery, analyse gene ontology (GO) terms and pathways associated with HL and expand upon the key genes that explain a significant proportion of HL in the Pakistani population.

| CHAR AC TERIS TIC S AND CLINIC AL CL A SS IFI C ATI ON S FOR HE ARING LOSS
HL, the permanent loss of hearing ability, encompasses several key characteristics. 3Firstly, the anatomical site of the cochlear defect is used to distinguish between conductive or sensorineural HL (SNHL), with the latter being most prevalent. 4Conductive HL occurs due to abnormalities in the outer or middle ear that hinder sound conduction, while SNHL results from damage to the inner ear or auditory nerve.When both conductive and sensorineural components are present, mixed HL ensues.Additionally, abnormalities or malfunctions beyond the cochlea, such as in the eighth cranial nerve, auditory brainstem, or cortex, lead to central auditory impairment. 5The second characteristic pertains to the age of onset relative to speech development. 3Pre-lingual HL manifests before the critical period for speech development, including congenital onset or HL already present at birth.Post-lingual HL, on the other hand, includes individuals with onset following the critical period for speech acquisition, spanning from childhood to advanced age and includes age-related HL. 3 The third feature relates to the degree of HL and the associated hearing thresholds used to define severity levels, which include mild (hearing threshold 20-40 dB), moderate (41-55 dB), moderately severe (56-70 dB), severe (71-90 dB) and profound (90 dB). 3 HL can affect low (<1000 Hz), mid (1000-2000 Hz) or high (>2000 Hz) frequencies.Classifying HL based on these factors is crucial for determining the most appropriate treatment and management strategies for each individual.The fourth attribute of HL concerns the presence or absence of progression, which is determined by serial audiometry.Although most late-onset HL typically follows a progressive course, the majority of individuals with congenital HL have a nonprogressive form.HL can affect one or both ears, referred to as unilateral or bilateral, respectively.Asymmetric HL is another possibility where both ears are affected to differing degrees. 6The sixth feature is whether HL is part of a syndrome that involves concomitant abnormalities in additional organ systems or is the sole presenting feature, as seen with non-syndromic HL (NSHL), which is observed in the vast majority of congenital cases. 3heritance patterns are another defining characteristic of HL.
Most hereditary HL exhibits an autosomal recessive inheritance, accounting for around 75% of congenital cases, 7 while roughly 18%, 1%-3% and 1% of NSHL are caused by autosomal dominant, X-linked and mitochondrial forms, respectively. 8Those with autosomal recessive non-syndromic hearing loss (ARNSHL) inherit either a single heterozygous variant from each asymptomatic parent, as observed for homozygous variants, or receive two distinct variants in the same gene, one from each parent, known as compound heterozygous variants.If both parents carry a single disease allele on the same gene, their offspring have a 25% chance of inheriting two disease alleles to develop HL, a 50% chance of inheriting one disease allele to be an asymptomatic carrier and a 25% chance of inheriting no disease alleles and being unaffected.

| ME THODS US ED FOR IDENTIFI C ATI ON OF ARN S HL G ENE S
Over the past three decades, there has been significant progress in identifying genes and loci associated with HL in Pakistani families (see Table 1 and Figure 1), using a range of evolving technologies.
One method used to locate Mendelian disease loci involves classic parametric linkage analysis, which utilizes genetic STRP markers or SNP maps along with information about mode of inheritance, penetrance and allele frequencies. 9This approach is based on the principle that chromosomal regions near genetic variants segregate together during meiosis.By analysing nearby markers, a genomic locus carrying a causative mutation may be statistically linked to the disease.The strength of the linkage evidence (or lack thereof) is assessed using the estimation of a logarithm of the odds (LOD) score.
A LOD score greater than 3.0 is considered significant for linkage. 10ce one or more potentially significant linked intervals have been TA B L E 1 Fifty-one ARNSHL genes were found mutated in the Pakistani population in the last 25 years listed in chronological order.Abbreviations: ES, exome sequencing; GS, genome sequencing; HM, homozygosity mapping; LA, linkage analysis; ND, not defined; NGS, next-generation sequencing.

TA B L E 1 (Continued)
identified through linkage analysis, the next step involves candidate gene sequencing of all genes in the region.Thirty-nine ARNSHL genes have been identified in the Pakistani population with the support of linkage analysis.Homozygosity mapping is considered the best method for uncovering chromosomal regions that may harbour a causal variant in consanguineous families. 11This method involves examining genotypes from multiple affected and unaffected family members to identify the number and size of runs of homozygosity.
This information can be used to narrow down the genomic regions that are likely to contain the disease-causing variant.In genetically heterogeneous disorders, homozygosity mapping can reduce the need to sequence various candidate genes and instead focus on specific genomic regions. 12Twelve ARNSHL genes have been identified by using homozygosity mapping.
Gene discovery has been further revolutionized by advances in exome and genome sequencing.Exome sequencing targets the protein-coding portion of genes, representing only about 1% (30 Mb) of the genome but capturing 85% of the disease-causing mutations in Mendelian disorders. 63Consequently, sequencing the entire exome has become a powerful tool for identifying diseaseassociated variants, including monogenic disorders like HL as well as predisposing variants in common diseases.This has led to faster identification of such variants. 64Genome sequencing provides a comprehensive view of an individual's entire genome, including both coding and non-coding regions, allowing for the identification of both rare and common variants associated with HL.In recent years, genome sequencing has enabled the discovery of new HL genes, particularly when variants reside in non-coding regions that were previously overlooked. 65Using exome and genome sequencing, 22 gene variants in novel NSHL-associated genes in the Pakistani population have been identified.Some of these 22 genes (12 genes) were also identified using other techniques described above such as linkage analysis and homozygosity mapping (as mentioned in Table 1).
Moreover, it has facilitated the identification of pathogenic variants in known genes and enables the assessment of structural variations, such as copy number variations and structural rearrangements that can contribute to HL.As sequencing technologies continue to advance and become more cost-effective, genome sequencing is likely to become an increasingly valuable tool for HL gene discovery and expedite the development of effective therapies.
In the Pakistani population, variants in a total of 51 genes have been implicated in ARNSHL through the approaches described above (Table 1).Owing to the unique genetic background and high consanguinity rate of the Pakistani population, 39 of these 51 genes have been discovered between 1997 and 2022 (Figure 1

| G ENE ONTOLOG IE S AND PATHWAYS OF RECURRENT ARN S HL G ENE S IN PAKIS TAN
By conducting molecular genetic studies on consanguineous pedigrees, researchers have been able to identify deleterious alleles in 51 out of the 68 genes linked to ARNSHL, thanks in part to participation of Pakistani families with HL. 66 Notably, among the 51 autosomal recessive genes we describe, nine genes (GJB2, TECTA, MYO6, ESPN, TMC1, TPRN, ELMOD3, TBC1D24 and PTPRQ) have To gain further insights into the functional roles of the 51 ARNSHL-associated genes, gene enrichment analysis and MCODE algorithm were employed using Metascape. 67The annotation, en- The 'Count' refers to the number of genes in our list of 51 genes associated with ARNSHL that are included in the given ontology term.To classify the genes associated with ARNSHL according to their function, we performed pathway and process enrichment and network analysis using Metascape (http:// metas cape.org).Using this approach, we identified the enrichment of GO terms and genes related to various biological pathways.
development of targeted therapies aimed at restoring normal function to disrupted pathways.

| RECURRENT G ENE S AND VARIANTS A SSO CIATED WITH HE ARING LOSS IN THE PAKIS TANI P OPUL ATION
Despite the extensive genetic heterogeneity in ARNSHL, a few genes account for over half of the cases.One large cohort study of Pakistani patients identified variants in six genes (GJB2, HGF, MYO7A, SLC26A4, CIB2 and TMC1) that collectively explained up to 57% of recessively inherited severe to profound HL. 66 Another cohort study found pathogenic or possibly deleterious mutations in GJB2, MYO7A, CDH23 and MYO15A in 13 out of 21 (61.9%)consanguineous Pakistani families with HL. 68 This part of the review aims to shed light on the most prevalent genes and variants in the Pakistani population.By providing valuable insights into the molecular genetics and epidemiology of ARNSHL, this information can be utilized to develop effective genetic screening and counselling programs for individuals and families affected by this condition and also develop personalized treatment strategies for the affected individuals.

| CDH23
CDH23 (DFNB12, USH1D) encodes cadherin 23, a crucial component of the extracellular filaments responsible for formation and function of the mechanosensory hair bundles of the inner ear. 69e hair bundle consists of stereocilia, hair-like projections that respond to vibration of the basilar membrane and stimulate hair cells by depolarization. 70CDH23 is expressed not only in the cochlear hair cells but also in the vestibular hair cells and the photoreceptor layer of the retina. 71In fact, CDH23 is one of the most highly expressed genes in the vestibular hair cells, where it plays a crucial role in maintaining their mechanical and electrophysiological properties.Different isoforms of CDH23 have tissue-specific functions.Biallelic variants disrupting CDH23 cause ARNSHL (DFNB12) and Usher syndrome type I (USH1D), characterized by congenital SNHL, vestibular dysfunction and early onset retinitis pigmentosa. 72Although CDH23-associated NSHL has traditionally been associated with congenital onset and profound severity, recent studies have revealed a broad phenotypic spectrum that includes individuals with biallelic variants presenting HL in the second to seventh decade of life, expanding the understanding of DFNB12. 73As one of the most common genetic causes of HL, many CDH23 pathogenic alleles have been identified in affected individuals of Pakistani ancestry, contributing to up to 5% of NSHL and USH1D 1 due to numerous pathogenic alleles of CDH23. 66The c.2968G>A, p.(Asp990Asn) and c.6133G>A, p.(Asp2045Asn) alleles are major causes of CDH23-associated deafness in Pakistan but the most commonly occurring variant of these genes is c.6050-9G>A (splicing). 66,74At least 28 additional CDH23 variants have been identified in different ethnic groups in Pakistan.However, so far no experimental evidence shows an impact on protein function. 66,74,75A comprehensive understanding of disease mechanisms in CDH23-associated disorders may significantly impact the diagnosis and management of HL and deaf-blindness worldwide.

| CIB2
Calcium-and integrin-binding protein 2 (CIB2) is a crucial component of the mechano-transduction (MET) process in cochlear hair cells, demonstrated by the complete abolishment of MET currents upon genetic disruption. 76,77MET is the process by which sound vibrations are converted into electrical signals that the brain can interpret as sound.CIB2 is an elongation factor-hand domain-containing protein that binds Ca 2+ ions and has been associated with various functions, including integrin signalling in platelets and skeletal muscle and autophagy, indicating its considerable functional flexibility. 78Although its clinical presentation seems to be limited to hearing deficits, typically manifesting as bilateral, profound, pre-lingual HL, CIB2 has been linked to a wide range of functions beyond hearing physiology.A brief association with Usher syndrome was eventually refuted. 41,79A large Pakistani cohort study has estimated the prevalence of CIB2associated HL as 8.6%. 66The homozygous recurrent c.272T>C, p.Phe91Ser (NM_006383.3)variant is the major cause of NSHL in the Pakistani community and has been identified in 81 families. 41Two additional rare variants have been identified in affected Pakistani individuals: c.192G>C, p.Glu64Asp and c.297C>G, p.Cys99Trp. 41

| CLDN14
CLDN14, also known as DFNB29, encodes the claudin-14 protein, an essential membrane protein forming tight junctions in the inner ear. 15These tight junctions are crucial for compartmentalizing the endolymphatic and perilymphatic fluid compartments, maintaining cell polarity, and regulating intercellular permeability to solutes, ions, and water. 80CLDN14 is expressed in the inner and outer hair cells, supporting cells, and Reissner's membrane. 81It plays a crucial role in maintaining ion homeostasis and calcium levels in the endolymph and perilymph fluids, which is essential for the MET process of cochlear hair cells. 82MET is initiated by the opening of cation channels near the tip of the stereocilia and requires ion homeostasis to maintain ion gradients for preservation of the endocochlear potential. 80While the endocochlear potential in Cldn14-null mice has been determined as normal, rapid degeneration of outer hair cells and progressive slower degeneration of IHCs is thought to be due to the compromised tight junction barrier. 83In Pakistani families, seven different variants of CLDN14 are responsible for 2%-3.3% of cases of profound deafness or moderate to severe HL 66,84 with the most prevalent variant being c.254T>A, p.Val85Asp (NM_001146079.2) identified in 21 families. 85It has been shown that a founder effect contributes to the recurrence of this variant. 85A modifier of CLDN14-associated HL has been proposed but remains uncharacterized.The discovery of CLDN14's role in regulating intercellular permeability in the inner ear highlights the importance of genetic testing for CLDN14 in individuals with HL in the moderate range.

| GJB2
Gap junctions play a crucial role in maintaining cochlear potassium homeostasis, which is essential for hearing.GJB2 encodes connexin 26 and is one of the most common causes of HL worldwide. 86Connexin 26 oligomerizes to form hexameric hemichannels called 'connexons' in the plasma membrane.The connexons of neighbouring cells come together to form gap junctions, which facilitate intercellular communication. 87Gap junctions can be homomeric or heteromeric, depending on whether they are made up of one or many different connexin proteins.This affects their ability to selectively permeate certain molecules and ions. 88Gap junctions allow ions, nutrients and signalling molecules with molecular weights up to 1200 Da to pass between cells. 89This intercellular communication is critical for the normal growth, function, and repair of the sensory epithelia of the inner ear.These processes that are disrupted through genetic mutation result in eventual cell death and HL.In the Pakistani population, the prevalence of GJB2-associated HL ranges from 6.1% to 53% 90 and it is the most common cause of NSHL in South Asia. 66Patients with biallelic pathogenic variants typically have congenital-onset HL, ranging from mild to profound severity, depending on the variant, and it can be progressive in roughly half of patients. 86Many pathogenic variants have been identified in GJB2, with the most common ones in the Pakistani population being c.231G>A, p.Trp77Ter, c.71G>A, p.Trp24Ter and c.35delG, p.Gly12ValfsTer2, (NM_004004.5). 1,7,68,91ong these variants, individuals homozygous for the c.35delG variant exhibit extreme phenotypic heterogeneity worldwide, ranging from mild HL to profound deafness.A founder effect has been attributed to its high prevalence.The GJB2 c.35delG variant causes a premature stop codon in exon 2 out of 2 total exons, leading to a loss of protein function. 92In Pakistan, except for one variant associated with moderate-to-severe HL, all others are associated with severe to profound deafness. 66To date, 29 pathogenic variants of GJB2 have been identified in the Pakistani population. 66

| HGF
HGF (DFNB39) encodes hepatocyte growth factor (HGF), which plays a crucial role in various biological processes, including cell growth, survival, differentiation and branching morphogenesis, with implications for neuronal survival and differentiation. 93HGF dosage in the inner ear must be precisely calibrated for normal hearing.
Transgenic mouse models of Hgf overexpression and deficiency present deafness resulting from the failure of neural crest cell migration to the intermediate cell layer of the stria vascularis, which causes thinning, reducing the endocochlear potential and hair cell loss. 33,93ile HGF-associated HL is relatively prevalent in the Pakistani population, explaining 6-8% of severe to profound pre-lingual HL, it is rarely implicated in the diagnosis of individuals with HL in other populations. 66In the Pakistani population, three HGF variants have been identified, including a synonymous variant that affects splicing and two deletions in a highly conserved region of intron 4, which is part of the 3′ untranslated region of a short HGF isoform. 75The most common variant in the Pakistani population is c.482+1986_1988delTGA (NM_000601.6) in intron 4. 33 Mice with the 10 bp deletion corresponding to c.482+1991_2000delGATGATGAAA in human intron 4 developed defects in the stria vascularis due to failure of neural crest cell migration during development, resulting in a significantly reduced endocochlear potential. 93HGF activates the MET receptormediated signalling pathway and mediates diverse downstream pathways, including those involved in the epithelial-mesenchymal transition and the development of neural crest-derived lineages.
Overall, deleterious variants in HGF can result in impaired cell migration and the development of neural crest-derived structures in the inner ear, leading to a reduced endocochlear potential and HL.

| MARVELD2
MARVELD2 (DFNB49) encodes a member of the marvel protein family called tricellulin that is concentrated at the tight junctions, forming part of the continuous intercellular barrier between epithelial cells.Tight junctions are multi-protein complexes preventing leakage of solutes and water, acting as a seal between epithelial cells. 94icellular tight junctions are present in epithelial cells between supporting and hair cells, cochlear supporting cells and marginal cells of the stria vascularis. 27Although MARVELD2 is ubiquitously expressed in epithelial junctions, only the inner ear appears to be affected by the genetic disruption, suggesting that genetic compensation may be present in other organ systems that are absent in the inner ear. 27allelic variants in the Occludin-ELL domain of MARVELD2 have been shown to cause HL in multiple Pakistani families. 95These variants explain between 1.5% and 2.4% of all HL in Pakistan to a moderate to profound degree.The most recurrent variant of MARVELD2 is c.1331+2T>C (NM_001038603.3), and at least seven additional rare disease-associated variants have also been reported in the Pakistani population. 1 Tricellulin is essential for maintaining the integrity and stability of epithelial cells and their junctions. 27The occluding-ELL domain of MARVELD2 may be necessary for the maintenance of tricellular junctions and the proper functioning of the inner ear. 96leterious variants in tricellulin lead to tight junction disorganization, resulting in severe to profound sensorineural HL. 27

| MYO7A
MYO7A (DFNB2) encodes an unconventional myosin that plays a crucial role in maintaining the mechanical stability of the hair bundle.The hair bundle is the sensory structure located on the surface of hair cells in the inner ear, acting as a mechanotransducer that transforms sound waves or orientation information into electrical signals, which the brain interprets (Houdusse & Titus, 2021). 97is is achieved through the transportation of extracellular stereocilia links along actin filaments 98 at the upper tip-link density and ankle link region of the stereocilia by MYO7A, which is essential for the cohesion of the hair bundle. 99MYO7A co-localizes with several other proteins at the upper tip-link density and ankle link region of the stereocilia, including CDH23, USH1C, USH1G, ADGRV1 and USH2A, which are integral for the proper functioning of the hair bundle (Houdusse & Titus, 2021). 97Moreover, independent of molecular trafficking, MYO7A exerts force at the upper tip-link density region and tensions the MET complex, further highlighting its important role in hearing. 100In addition to its role in the inner ear, MYO7A is also expressed in retinal pigment epithelial cells where it is required for functional RPE65, a key protein in the retinoid cycle. 101MYO7A is also associated with Usher syndrome type I, an autosomal recessive condition characterized by deafness, vestibular impairment and retinitis pigmentosa.Biallelic variants in MYO7A account for 29% to 50% of all USH1 cases globally, making it the most common cause of this condition. 102Moreover, at least 11% of moderate to severe pre-lingual SNHL in the Pakistani population is due to MYO7A disruption 42 with at least 59 variants reported. 66Among these variants, c.397dupC, p.His133ProfsTer7 and c.470G>A, p.Ser157Asn (NM_000260.3) are the most prevalent in the Pakistani population. 68These variants exhibit founder effects that are not seen in other populations. 68

| MYO15A
Myosin XVa, a product of MYO15A, belongs to the unconventional myosin superfamily and is essential for the elongation of stereocilia in cochlear sensory and vestibular hair cells.The growth of the hair bundle is facilitated by the transportation of whirlin and Eps8 to the tip of stereocilia to form the stereocilia tip complex by Myosin XVa, which can help in the conversion of microvilli into fully mature stereocilia. 103MYO15A is involved in the regulation of actin and the transportation of elongation complexes at the distal stereocilia tip as well as other cargoes for actin. 99Notably, the motor and tail domains of myosin XVa have been identified as crucial for normal auditory structure and function. 104In Pakistani families, at least 49 MYO15A variants have been linked to 5-13% of progressive severe to profound bilateral SNHL. 66The majority of the variants in this gene have been found in affected individuals from one to three Pakistani families, except for two variants, c.6589C>T, p.Gln2197Ter and c.8158G>C, p.Asp2720His (NM_016239.3)found in deaf members of four families each. 59MYO15A variants significantly impact the motor domain, leading to dysfunction causing shorter stereocilia with an ectopic staircase structure, a condition associated with severe deafness. 105

| MSRB3
MSRB3 (DFNB74) encodes methionine sulfoxide reductase B3 (MSRB3) that plays a critical role in repairing oxidatively damaged proteins by catalysing the stereo-specific reduction of methionine-R-sulfoxides to methionine. 37Msrb3 localizes to the base of the stereocilia on the apical surface of hair cells. 106udies on the Msrb3 −/− mouse model have shown that MSRB3 is essential for the maturation and/or maintenance of stereociliary bundles since these mice exhibit progressive degeneration of the stereociliary bundles and apoptosis of hair cells. 106Moreover, genetic disruption of Msrb3 is more likely to be due to degeneration rather than abnormal development, since the hair cells of KO mice develop normally and have functional mechanotransduction channels until at least P3. 106The c. Lebanese families by a candidate gene approach. 111Since then, at least 23 OTOF variants associated with moderate-to-severe or profound HL have been identified in hearing-impaired individuals from Pakistan. 32,84Among these, one of the most common is the c.2122C>T, p.Arg708Ter 66 variant. 112This variant has been detected in both homozygous and compound heterozygous states in individuals with prelingual NSHL or auditory neuropathy in a large population cohort. 32

| SLC26A4
SLC26A4, also known as pendrin, is a protein that belongs to the solute carrier family 26 and functions as an anion exchanger, transporting negatively charged ions such as chloride, iodide and bicarbonate across cell membranes.This protein is crucial for the development of the cochlea and vestibular duct's bony snail shape structure. 113Variants in SLC26A4 (DFNB4) have been linked to both ARNSHL and Pendred syndrome. 114While individuals with isolated HL may experience bilateral, profound SNHL, those with Pendred syndrome may have concomitant HL, enlarged vestibular aqueduct and abnormal iodine organification. 115Approximately 12.4% of HL cases in the Pakistani community can be attributed to SLC26A4. 66While the majority of the identified variants are described as non-syndromic, especially in young individuals, the presence of Pendred syndrome may be frequently overlooked.for their recurrence. 84,116

| TMC1
Transmembrane channel-like protein isoform-1 (TMC1) plays a crucial role in the auditory system as it forms an ion-conducting pore of the MET channel in auditory hair cells. 117 ants account for 6.4% of recessively inherited HL cases. 66The most frequently occurring TMC1 variant in the Pakistani population is c.100C>T, p.Arg34Ter (NM_138691.2),which has been reported in two studies. 84,121This common pathogenic nonsense variant is a likely founder mutation in North African and Middle Eastern populations. 7,17

| TMPRSS3
Genetic mutation of type II transmembrane serine protease (TMPRSS3) causes variable HL.TMPRSS3 has four functional domains that include an N-terminal transmembrane domain, a low-density lipoprotein receptor A domain, a scavenger receptor cysteine-rich domain and a C-terminal serine protease domain. 122TMPRSS3 is expressed in various components of the developing inner ear, such as the stria vascularis, spiral ganglion neurons, 123 IHCs and cochlear aqueduct of the foetal cochlea and is critical for their normal development and maintenance 124 Additionally, cytoplasmic domains of TMPRSS3 raise the possibility that they may participate in intracellular signal transduction. 122Biallelic variants in TMPRSS3 are known for causing different types of HL with variable onset.DFNB10 is characterized by congenital or childhood-onset bilateral profound HL, 125 whereas DFNB8 is associated with a milder postlingual progressive HL, 126 both of which are caused by variants in TMPRSS3 (DFNB8/10).In Pakistan, TMPRSS3 variants are associated with stable, moderate-to-severe or profound HL and contribute up to 4% of the prevalence of HL. 66 Other frequently occurring variants include c.323-6G>A (splicing) and c.1219T>C, p.Cys407Arg. 84The recurring homozygous c.1219T>C, p.Cys407Arg (NM_024022.2) variant has been identified in 20 Pakistani families. 127Functional studies show that the variant protein has defective protease activity compared to the wild-type (Lee et al., 2003) as well as a failure to undergo proteolytic cleavage and activate the epithelial sodium channel (ENaC) in vitro. 124,128In silico analysis also supports that this missense variant has a negative effect on protein structure/function.There are 14 other rare variants in this gene that contribute to NSHL in the Pakistani population.

| CON CLUS ION
This review explores the clinical and genetic complexities of HL, defining and describing key characteristics, terminology and genetics.
), underscoring the significant contribution of the Pakistani population to unravelling the genetics of HL.Although next-generation sequencing-based molecular tests are still in their infancy, they have shown clinical value for singlegene diseases.The full potential of exome and genome sequencing will enrich genomic medicine beyond rare single-gene disorders.Further developments in next-generation sequencing technologies and bioinformatics tools will enhance data analysis and clinical extraction.Recent studies justify the effort and expense of incorporating these innovative ideas into molecular diagnostics practice.While advances in next-generation sequencing have sped up the detection of disease-causing variants, they cannot overcome universal limitations faced in Mendelian disorders.Explorationof the genetics of ARNSHL is optimal with large pedigrees that ideally support gene mapping approaches and next-generation sequencing.Furthermore, identification of variants in compound heterozygosity is critical for validating genetic findings.Variants with limited evidence to conclusively assign likely pathogenic or likely benign classifications remain a great challenge in genomic medicine in general.Variable expressivity can affect interpretation as well as outcomes, as not everyone who harbours particular variants will develop the disease due to possible incomplete penetrance.It is critical to use these technologies in collaboration with various patient populations, genetic counsellors, and medical geneticists.This collaborative endeavour will be crucial for increasing our understanding of the genetic basis of HL and discovering novel therapies to treat it.

F I G U R E 1
Timeline of 39 ARNSHL genes identified by collaborating with the Pakistani population over the past 25 years.been associated with an autosomal-dominant inheritance pattern as well.While the identification of 51 genes associated with HL may seem daunting, researchers can simplify the complexity by examining the established metabolic and cell signalling pathways through which these genes interact in various organs or by identifying enriched ontology clusters.One of the fundamental presumptions of network analysis and ontology clustering is that there are functional connections between genes whose malfunction leads to disease manifestation.Understanding biological systems, disease states and the mechanisms by which drugs affect them relies on identifying these common pathways and ontology terms.Therefore, by recognizing the shared biological pathways involved in HL and other disorders, researchers can gain insights into the underlying causes of these diseases and develop effective therapeutic interventions.
richment and genes used to perform the Metascape analysis are shown in TableS1.Enrichment analysis using Metascape identified four areas of enriched protein clusters where proteins are closely related based on similar pathways involved.The first cluster of genes was involved in sensory perception of sound (27 genes, GO:0007605), the second cluster in sensory processing of sound by cochlear inner hair cells (IHCs) (19 genes, R-HSA-9662360), the third cluster in actin cytoskeleton organization (7 genes, GO:0030036), and the fourth cluster in retina homeostasis (6 genes, GO:0001895) (Figure2).To facilitate the visualization of the results, we created a pie chart using Microsoft Excel software to illustrate the count of genes belonging to each category of enriched GO terms.Protein clustering also revealed that the 51 genes associated with ARNSHL regulate several common biological pathways.These pathways include cell-cell junction organization (5 genes, GO:0045216), cellular response to chemical stress (5 genes, GO:0062197) and regulation of plasma membrane-bounded cell projection organization (5 genes, GO:0120035).Other biological processes involved were monoatomic cation homeostasis (4 genes, GO:0055080), endocytosis (4 genes, GO:0006897), cellular response to hormone stimulus (4 genes, GO:0032870), endothelial cell development (3 genes, GO:0001885) and MET activates PTPN11 (3 genes, R-HSA-8865999) (Figure2).'MET activates PTPN11' refers to a signalling pathway that involves the interaction of two proteins of MET and PTPN11.This pathway has been implicated in a variety of cellular processes and diseases, including cancer and developmental disorders.Overall, our bioinformatics analysis highlights the intricate role of ARNSHL genes in various cellular signalling pathways and biological processes.These findings have important implications for understanding the underlying mechanisms of ARNSHL and for the F I G U R E 2 Pie chart showing the results of Metascape analysis of enriched gene ontology (GO) clusters.The chart displays the top 12 clusters and their representative enriched terms (one per cluster).

SLC26A4
has several recurring variations.In the majority of affected individuals, HL is associated with three variants: c.269C>T, p.(Ser90Leu), c.716 T > A, p.(Val239Asp), and c.1337A > G, p.(Gln446Arg) (NM_000441.1).A founder effect has been shown Over the past 5 years, TMC1 has emerged as a leading contender for the MET channel in auditory hair cells of the inner ear.Hair cells convert acoustic and vestibular stimuli into electrical responses through the activation of MET. 118TMC1 is thought to have a six-transmembrane domain structure similar to several other ion-channel subunits and is transported to the tips of the stereocilia in the sensory hair bundle where the MET channel is located. 119TMC1 variants associated with human deafness result in loss of typical MET currents and hair cell senescence, causing cell death.Valuable insights into the pathogenesis of TMC1-associated deafness have been gained from studies of mutant mice. 120Variants in TMC1 cause both dominant (DFNA36) and recessive (DFNB7/11) forms of NSHL.Both progressive postlingual and pre-lingual profound HL have been associated with TMC1 variants.In Pakistan, at least 32 different TMC1 vari- After 25 years of engaging the Pakistani population, researchers have uncovered variants in 51 genes and made significant progress in understanding the most frequently implicated ARNSHL genes.Of these, 39 genes causally related to HL were discovered through the use of gene mapping methodologies and sequencing strategies in consanguineous Pakistani families.This aggregated knowledge has established 13 being the most commonly involved in the molecular diagnosis of Pakistani patients (CDH23, CIB2, CLDN14, GJB2, HGF, MARVELD2, MYO7A, MYO15A, MSRB3, OTOF, SLC26A4, TMC1 and TMPRSS3).We detailed their purpose and highlighted important variants from the Pakistani community's standpoint.For a detailed molecular understanding of these ARNSHL genes, we also categorized enriched GO terms and shared pathways using Metascape.Engaging the Pakistani community has been fundamental in advancing gene discovery.The high prevalence of consanguinity and congenital HL, a predominantly recessive trait, has provided